Dopaminergic Neurons Linking Threat Processing to Cardiac Modulation and Locomotor Responses
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eLife Assessment
This important study identifies two pairs of dopaminergic neurons (DA-WED) in Drosophila that coordinate cardiac deceleration and locomotor responses to a mechanical threat. The evidence is convincing, supported by comprehensive optogenetic, physiological, and behavioral experiments showing that these neurons are required for and sufficient to drive threat-associated cardiac slowing. The proposed role of cardiac deceleration as an interoceptive contributor to locomotion is intriguing, but should be presented more cautiously, as the causal relationship between heartbeat changes and locomotor output remains less directly established. The work will be of broad interest to those interested in neural circuits, neuromodulation, and the integration of physiological and behavioral responses.
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Abstract
Heartbeat and behavior are tightly coordinated during defensive states, yet the neuronal mechanisms linking threat processing to cardiac modulation—and the potential contribution of cardiac dynamics to behavioral output—remain poorly understood. Here we show in Drosophila that mechanical threat triggers locomotion together with cardiac deceleration. We identify two dopaminergic neurons, termed DA-WED neurons, that mediate this cardiac response: silencing these neurons markedly attenuates threat-induced cardiac deceleration, whereas optogenetic activation induces cardiac deceleration in the absence of threat. Calcium imaging further shows that DA-WED neurons are activated by mechanical threat. Linking cardiac dynamics to behavior, direct optogenetic manipulation of cardiomyocytes that quantitatively reproduces threat-evoked cardiac deceleration is accompanied by increased locomotion. Together, these results identify a dopaminergic descending pathway that links threat processing to cardiac modulation and suggest that cardiac dynamics may contribute to shaping defensive behavior.
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eLife Assessment
This important study identifies two pairs of dopaminergic neurons (DA-WED) in Drosophila that coordinate cardiac deceleration and locomotor responses to a mechanical threat. The evidence is convincing, supported by comprehensive optogenetic, physiological, and behavioral experiments showing that these neurons are required for and sufficient to drive threat-associated cardiac slowing. The proposed role of cardiac deceleration as an interoceptive contributor to locomotion is intriguing, but should be presented more cautiously, as the causal relationship between heartbeat changes and locomotor output remains less directly established. The work will be of broad interest to those interested in neural circuits, neuromodulation, and the integration of physiological and behavioral responses.
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Reviewer #1 (Public review):
Summary:
This study by Tsuji et al. explores a mechanical threat model in Drosophila using air puffs as a stimulus. The authors first establish the paradigm and show that air puffs induce cardiac deceleration along with increased locomotion. They then identify dopamine as a key regulator of this response and go on to map the underlying circuit. In doing so, they pinpoint two pairs of DA-WED neurons as critical players. They carefully used intersectional strategies to achieve relatively clean labeling of these neurons, which helps ensure that the observed effects can be attributed specifically to DA-WED neurons. They further show that DA-WED neurons are both required and sufficient to drive cardiac deceleration, and that their activity increases in response to air puff stimulation. These neurons also …
Reviewer #1 (Public review):
Summary:
This study by Tsuji et al. explores a mechanical threat model in Drosophila using air puffs as a stimulus. The authors first establish the paradigm and show that air puffs induce cardiac deceleration along with increased locomotion. They then identify dopamine as a key regulator of this response and go on to map the underlying circuit. In doing so, they pinpoint two pairs of DA-WED neurons as critical players. They carefully used intersectional strategies to achieve relatively clean labeling of these neurons, which helps ensure that the observed effects can be attributed specifically to DA-WED neurons. They further show that DA-WED neurons are both required and sufficient to drive cardiac deceleration, and that their activity increases in response to air puff stimulation. These neurons also contribute to the locomotor response. Directly inducing cardiac deceleration via optogenetic manipulation of cardiomyocytes also increases locomotion, suggesting a link between cardiac state and behavioral output.
Strengths:
Overall, the experiments are thoughtfully designed, well-controlled, and clearly presented. The figures are easy to follow, and the conclusions are generally well supported by the data. The manuscript is also clearly written, with a discussion that acknowledges potential caveats and outlines future directions. The genetic tools, behavioral paradigm, heart rate measurement approaches, and stimulation methods introduced here will be valuable resources for the community.
Weaknesses:
A few minor points to add to the clarity of the manuscript:
(1) The DA-WED driver (R48A08-AD ∩ VT008692-DBD ∩ TH-FLP) appears quite clean in the brain. However, since the study focuses on cardiac function and locomotion, it would be helpful to check expression in cardiomyocytes and the ventral nerve cord. This would help rule out any off-target expression that might contribute to the phenotypes and further support the idea of a descending pathway from brain dopaminergic neurons.
(2) Since DA-WED>Kir2.1 abolishes the puff-induced locomotor response (Figure 5b), suggesting that DA-WED neurons are directly involved in mediating locomotion. In the model (Figure 5L), it might make more sense for the pathway from mechanical threat to locomotion to pass through DA-WED neurons. The authors could consider adjusting the schematic if they agree.
(3) In line 408, Figure 5K should be 5L as it's a discussion of the model.
(4) In Figure 5j, the x-axis is missing time labels. Even if it matches Figure 5h, adding labels would make it easier to interpret at a glance.
(5) In line 312, it would be helpful to briefly explain why a 28 ms light pulse was used, compared to other pulse durations elsewhere in the paper.
(6) The cardiac deceleration seems to recover quickly after the air puff ends, whereas the locomotor response persists longer (around 10-15 seconds; see Figure 1 and Figure 5). This difference might suggest that DA-WED neurons influence locomotion through an additional or partially independent pathway, beyond their role in cardiac regulation. It could be worth briefly discussing this possibility.
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Reviewer #2 (Public review):
Summary:
The authors study cardiac deceleration during threat responses in Drosophila. Particularly, it focuses on identifying the neuronal control of this deceleration. Using behavioral and cardiac tracking and analysis, genetics, and calcium imaging, they identify two pairs of dopaminergic neurons involved in cardiac deceleration during air puff responses
Strengths:
The study is overall well done, and the paper is clearly written. Particularly, the work on identifying the two pairs of dopaminergic neurons involved in cardiac deceleration using a series of drivers and generating new ones is rigorous and extensive. Finally, the authors manipulate the heartbeat to investigate how it influences threat responses
Weaknesses:
There are, however, several points that need to be clarified, as some claims are not …
Reviewer #2 (Public review):
Summary:
The authors study cardiac deceleration during threat responses in Drosophila. Particularly, it focuses on identifying the neuronal control of this deceleration. Using behavioral and cardiac tracking and analysis, genetics, and calcium imaging, they identify two pairs of dopaminergic neurons involved in cardiac deceleration during air puff responses
Strengths:
The study is overall well done, and the paper is clearly written. Particularly, the work on identifying the two pairs of dopaminergic neurons involved in cardiac deceleration using a series of drivers and generating new ones is rigorous and extensive. Finally, the authors manipulate the heartbeat to investigate how it influences threat responses
Weaknesses:
There are, however, several points that need to be clarified, as some claims are not entirely supported by evidence.
The authors, for example, claim that dopaminergic neurons are responsible for cardiac deceleration (during the air puff, lines 182-3, page 9). However, based on the work in this study, it seems that other neurons could be involved in this control as well. In addition to dopaminergic neurons, the authors test serotonergic and octopaminergic neurons, which, based on silencing experiments, also show an implication in heart-beat deceleration. Furthermore, because they find that dopaminergic neurons are the only ones that, upon thermogenetic activation, lead to lower heart beat frequency, they conclude that the dopaminergic neurons are responsible for air -puff induced cardiac deceleration.
However, these activation experiments are done in a different context than the air puff experiments (at a higher temperature, which could have an effect on the heartbeat changes upon activation of different neuron groups), and because silencing of other monoaminergic neuron types during the air puff also resulted in less cardiac deceleration, one cannot exclude the implication of octopaminergic or serotonergic neurons in air-puff-induced deceleration.
Activation experiments without high temperatures (using, for example, optogenetics) and/or in the presence of the air puff would be important to determine that the dopaminergic neurons are the main type of monoaminergic neurons involved in air-puff-induced cardiac deceleration. Otherwise, the related claims should be rephrased in a way that clearly doesn't exclude a possible implication of other monoaminergic neurons.
Regarding the interactions between the cardiac deceleration and locomotion, the authors propose, based on the results, that the optogenetic cardiac deceleration is sufficient to induce an increase in locomotion, and that it is the decrease in heartbeat that would be responsible via interoceptive pathways to trigger an increase in locomotion. In the model they propose, the DA-WED neurons would induce a decrease in heartbeat that, in turn, would trigger an increase in locomotion. There is not enough proof that cardiac deceleration is the one that triggers an increase in locomotion during air puff responses. As the authors themselves state, the experiments that would demonstrate this would involve preventing cardiac deceleration while optogenetically activating DA-WED. It can therefore not be excluded that the DA-WED neurons trigger an increase in locomotion that is possibly modulated by the cardiac activity. Both alternatives should be considered (models in Figures 4 and 5).
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Reviewer #3 (Public review):
Summary:
In this elegant study, Tsuji et al. identify a relationship in Drosophila between cardiodynamics and threatening stimuli where mild air puffs elicit a brief bradycardia that coincides with locomotion increases. They then take advantage of the arsenal of genetic tools available in the fruit fly to reveal the indispensability of dopamine, through the action of Dop1R2, in this phenomenon. Further, they pinpoint the source of this dopamine to two specific pairs of neurons - DA-WED that are threat-activated. They then test and find a potential role for cardiac interoception from the heart in linking behavior and cardiodynamics.
Strengths:
This is an interesting and timely story that brings together the tools of fruit fly systems neuroscience and links it with physiology. The experiments are well done and …
Reviewer #3 (Public review):
Summary:
In this elegant study, Tsuji et al. identify a relationship in Drosophila between cardiodynamics and threatening stimuli where mild air puffs elicit a brief bradycardia that coincides with locomotion increases. They then take advantage of the arsenal of genetic tools available in the fruit fly to reveal the indispensability of dopamine, through the action of Dop1R2, in this phenomenon. Further, they pinpoint the source of this dopamine to two specific pairs of neurons - DA-WED that are threat-activated. They then test and find a potential role for cardiac interoception from the heart in linking behavior and cardiodynamics.
Strengths:
This is an interesting and timely story that brings together the tools of fruit fly systems neuroscience and links it with physiology. The experiments are well done and tell a very nice story. In particular, the primary message of the paper - that the authors have identified specific dopaminergic neurons that regulate cardiac activity - is sound.
Weaknesses:
There are no important problems with the scientific approach. Rather, there are some interpretive changes I would consider.
(1) The changes in heart rate are small (10% or so), and, as far as I can tell, are evident for a beat or two. So the data may be better interpreted not as a change in rate but as a lengthening of diastole for a beat or two. That may seem a petty difference, but it might point to particular stretch-activated systems or changes in blood flow as the determinant.
Heart rate must be averaged over time, and so might be blurring the effects. It may be useful to produce figures centered on beat count and duration rather than time. Because the effect may even be just on a single beat, we suggest the authors try plotting the average beat duration for each beat that follows the air puff. If it's really just the first beat, using a quantification of the change of this duration relative to the average that precedes the puff may produce more striking figures.
(2) The author's model that cardiac deceleration leads to walking data is only partially supported by their data. In the first figure, the relationship between cardiac deceleration and walking probability seems to be inverted relative to their model (weak stimulus -> strong cardiac effect and weak locomotor effect; strong stimulus-> weak cardiac effect and strong locomotor effect). It is possible that this discrepancy may disappear when the authors look at beat duration rather than heart rate (for instance, if following the strong stimulus, there is a very long beat that is followed by tachycardia, thus weakening their observed HR change). It would also be easier to relate this data in Figure 1 to their interoceptive model if some data were shown that illustrated the relative timing of the cardiac change and the locomotor start.
(3) Also, since the locomotor and cardiac changes are probabilistic, it would be very useful to see how their respective probabilities change when conditioned on the other. According to their interoceptive model, locomotion should preferentially increase on trials where cardiac deceleration occurs. The authors should discuss this incongruity and also potential alternative interpretations of their cardiac manipulation experiments. Perhaps the bradycardia makes them more sensitive to threats - as suggested in the introduction? Control flies show a mild increase in locomotion following green light (Figure 5j), so perhaps by slowing the heart, they are more sensitive and thus respond more strongly to this stimulus?
(4) Looking at the example shapes of the beats in Figure 5g versus Figure 1c, the optogenetically induced diastole has a very different shape from the naturally occurring long beat. Thus, the exact cardiac stimulus may be unnatural. If this is true across trials and animals, it may be worth considering that the funny beat (like an anxiogenic atrial fibrillation in mammals) is the source of the fear and, in turn, locomotor behavior (also interesting!) rather than being a true replication of the cardiac events seen following the puff stimulus.
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